Construction Of RNA-cleaving DNAzyme Based Fluorescent Probes And Their Applications In The Detection Of Tumor Markers

Tumor markers are substances that are directly produced by tumor cells or induced by the body during the process of tumor occurrence and proliferation,such as certain proteins and miRNAs.More and more studies have shown that the content of tumor markers is closely related to the degree of cancer development.Therefore,the development of highly sensitive and specific tumor marker detection methods is of great significance for the diagnosis and subsequent treatment of clinical diseases.With the development of tumor marker research,the methods and technologies for tumor marker detection are constantly updated.RNA-cleaving DNAzyme is a synthetic DNA enzyme.Because of its catalytic activity that mimics protease,mild reaction conditions,and good biocompatibility,it is widely used in the detection of various tumor markers.However,most of the RNA-cleaving DNAzyme-based probes currently reported often have problems such as low sensitivity,poor temporal and spatial controllability,and high false positive signals.Therefore,there is an urgent need to develop high-efficiency RNA-cleaving DNAzyme probes to realize the time and space controllable high-precision detection of tumor markers.Based on the signal amplification ability of RNA-cleaving DNAzyme and combined with nanotechnology,aiming at several important tumor markers,three RNA-cleaving DNAzyme fluorescent probes are designed for the detection and imaging of tumor exosomes,telomerase RNA that regulates chromosome length in cells,and miR-21,which is involved in cell differentiation and apoptosis,respectively.The details are as follows:(1)A fluorescent probes based on aptazyme using for the highly sensitive detection of exosomes in clinical serum samplesCombining the signal amplification ability of DNAzyme and the specific recognition performance of aptamer,this work designed a hairpin aptazyme for the detection of exosomes with high sensitivity and specificity.When encountering the target exosomes,the aptamer can specifically bind to the PTK7 protein on the surface of the exosomes,which leads to the structural conversion of the aptazyme,the catalytic activity of the DNAzyme is activated,and then the molecular beacon is cleaved circularly,thereby realize the recovery of fluorescence and the amplified detection of exosomes.Under the best conditions,the linear detection range of this method is 5×103-2×106 particles/μL,and the limit of detection(LOD)is as low as 3.5× 103 particles/μL(3σ).In addition,the probe has been successfully used in the detection of target exosomes in clinical blood samples,achieving high-specific discrimination between cancer patients and healthy individuals.(2)A DNAzyme-based light-controlled sensing method for imaging research of telomerase RNA in living cellsOn one hand,DNAzyme fluorescent probes will have a certain background interference in the biological environment,on the other hand,the design of using exogenous ultraviolet light to activate DNAzyme fluorescent probes to realize the controllable detection of target objects in time and space has attracted increasing attention.Therefore,this work intends to use manganese dioxide(MnO2)nanosheets as a carrier to construct a fluorescent probe of DNAzyme activated by ultraviolet light for the detection of intracellular telomerase RNA.First,the nucleic acid probe is transported into the cell through the MnO2 nanosheet.The MnO2 nanosheet is lysed under the reduction of the glutathione(GSH)with a high concentration in the cell,releasing Mn2+ and the probe.The probe is activated by ultraviolet light at 365 nm in the first step,a strand displacement reaction occurs in the presence of the target telomerase RNA,and then the catalytic activity of the DNAzyme is activated in the second step.With the assistance of Mn2+,DNAzyme cyclically cut the molecular beacon substrate chain,which causes the fluorophore to move away from the quencher and restore the fluorescence,thereby realizing the amplified detection of telomerase RNA in living cells.The experimental results show that the probe can detect and image telomerase RNA spatiotemporally in living cells.(3)The research on the controllable construction and application of DNA nanowalking machine based on DNAzymeAlthough traditional DNA molecular motors based on gold nanoparticles have the advantages of rapid and efficient detection of targets,their Au-S bonds are prone to breakage under the action of the thiols with high concentrations in the cell microenvironment,which can easily lead to false positive signals,and the number of probes in the surface of gold nanoparticles is also difficult to control.Based on this,this experiment designed a dendritic DNA polymer gradually assembled from Y-type DNA.Through the rational design of bases,different ratios of DNAzyme chains and substrate chains can be modified on its surface to obtain the best response performance.And because of the stability of its nanostructure,it can remain stable under the thiols with high concentrations in the cell.When the target miRNA is present,the miRNA will activate the DNAzyme to realize the cyclic cutting of the substrate chain,like a nano-walking machine walking along the surface of the dendritic DNA polymer,so as to realize the amplified detection of the miRNA in the cell.Experimental results show that the nano-walking machine has high biological stability and can achieve highly sensitive detection of targets.